The present invention relates to compositions and methods for modulating serum cholesterol. In one aspect, the invention features novel anti-lipemic drugs that include at least one identified effector of the Low Density Lipoprotein (LDL) receptor and at least one identified serum cholesterol inhibitor. In a particular aspect, the anti-lipemic drug includes at a sphingolipid or protein modifying same linked to the serum cholesterol inhibitor. Additionally provided are methods for using the anti-lipemic drugs to significantly stabilize or reduce serum cholesterol levels in a subject mammal and particularly a human patient.
There is nearly universal agreement that cholesterol is a key lipid constituent of cell membranes. Cholesterol is generally understood to be essential for normal growth and viability of most higher organisms. Too much serum cholesterol has been correlated with life threatening lipid related diseases including hyperlipoproteinemia, stoke, coronary heart disease, and especially atherosclerosis and related conditions. See generally Stryer, L. (1988) in Biochemistry, 3rd Ed. W. H. Freeman and Co. New York, pp. 547-574; and Brown, M. S. and Goldstein, J. L. (1993) in The Pharmacological Basis of Therapeutics (8th Ed.) Gilman, A. G. et al. eds. McGraw-Hill/New York, pp. 874-896.
The regulation of serum cholesterol in mammals and particularly primates has attracted significant attention. It is often reported that regulation of cholesterol homeostasis in humans and other mammals involves regulation of cholesterol production, bile acid biosynthesis and catabolism of specific serum cholesterol carriers. Important serum cholesterol carriers are called LDL (low density lipoprotein) particles. The LDL receptor has been reported to facilitate internalization of the LDL particle into those cells in need of cholesterol. See e.g., Brown, M. S. and Goldstein, J. L. (1986) Science 232: 34-47; and Goldstein, J. L. and Brown, (1986) Nature, 348: 425; and references cited therein.
The LDL receptor has been disclosed as impacting serum cholesterol levels in humans. For example, there has been recognition that cells with enough cholesterol do not make sufficient LDL receptors, thereby reducing or even blocking uptake of cholesterol by the cell. In this instance, serum cholesterol levels rise substantially which can contribute to the development or severity of disease. Conversely, cells in need of cholesterol often have capacity to make more LDL receptors, thereby facilitating a decrease in serum cholesterol. Accordingly, there has been specific attention focused on regulating the LDL receptor as one therapeutic approach for stabilizing or reducing serum cholesterol levels in human patients.
In particular, it has been reported that transcription of the LDL receptor gene is suppressed when sterols accumulate and induced when sterols are depleted. Sterol sensitivity is thought to be conferred by a 10 basepair (bp) sequence upstream of the LDLr gene known as the sterol regulatory element (SRE). It has been disclosed that the mature form of the sterol regulatory element binding protein-1 (SREBP-1) binds to the SRE and promotes transcription.
There have been additional reports that the activity of SREBP-1 is influenced by sterol induced proteolysis. There is recognition that the SREBP-1 proteolysis is impacted in some settings by a cell receptor termed xe2x80x9ccytokine tumor necrosis factorxe2x80x9d (TNF-xcex1).
In particular, the TNF-xcex1 receptor has been reported to influence a wide range of biological effects. However, the TNF-xcex1 receptor remains incompletely characterized. Elucidation of TNF-xcex1 pathways is sometimes complicated by presence of at least two TNF receptors. The receptors share some common downstream effectors but also signal via receptor specific pathways. See the references cited below for additional disclosure relating to the TNF-xcex1 receptor.
There has been understanding that one consequence of TNF signaling is the activation of neutral sphingomyelinase (N-SMase). Neutral sphingomyelinase is a membrane bound enzyme that catalyzes the hydrolysis of sphingomyelin to ceramide and phosphocholine at a pH optima of 7.4. The role of neutral sphingomyelinase in signal transduction has primarily been related to ability to generate the lipid second messenger ceramide. In addition to TNF-xcex1, Fas receptor ligand, vitamin D3, interleukin-1xcex2, nerve growth factor, anti-CD28 antibodies and xcex3-interferon have all been shown to increase ceramide levels.
In particular, sphingomyelinases type-C (E.C. 3.1.4.12) are a group of phospholipases that catalyze the hydrolytic cleavage of sphingomyelin via the following reaction (1).
Sphingomyelinxe2x86x92Ceramide+Phosphocholinexe2x80x83xe2x80x83(1)
See S. Chatterjee, Adv. Lipid Res., 26:25-48 (1993); S. Chatterjee et al., J. Biol. Chem., 264:12,534-12,561 (1989); and S. Chatterjee et al., Methods in Enzymology, Phospholipase, 197:540-547 (1991).
In addition to the biological roles of sphingomyelin and ceramide in signal transduction pathways involving cell regulation, more recent evidence has emerged suggesting that sphingomyelinases may be involved in cholesterol homeostasis and particularly induction of LDL receptor activity. See S. Chatterjee, Advances in Lipid Research, 26:25-48 (1993). Additional work supports a possible role of ceramide in programmed cell death and/or xe2x80x9capoptosisxe2x80x9d and activation of the gene for nuclear factor (NF)-kB. See A. Alessenko and S. Chatterjee, Mol. Cell. Biochem., 143:169 (1995).
It has been suggested that certain enzymes involved in making cholesterol exert a significant effect on cholesterol homeostasis. Accordingly, there has been substantial interest in identifying drugs with capacity to modulate these enzymes especially to stabilize or reduce serum cholesterol to tolerable ranges. Illustrative agents include commercially available serum cholesterol inhibitors such as fluvastatin, simvastatin, lovastatin, pravastatin, and atorvastatin. See Brown, M. S. and Goldstein, J. L. (1993), supra for additional disclosure relating to these and other agents such as mevinolin (compactin).
Although some clinical benefit has been reported to follow use of these and other serum lowering agents, there have been reports of significant side-effects. See e.g., Brown, M. S. and Goldstein, J. L. (1993), supra; and Physicians"" Desk Reference 1997 (515st ed.) Medical Economics Co. Accordingly, there is a need to have drugs that exhibit more desirable characteristics such as enhanced potency and better patient tolerance. There is a specific need to reduce levels of administered cholesterol lowering agents for some patients.
There is also a need to identify drugs that can modulate the SREBP-1 protein and especially the LDL receptor. Moreover, methods for identifying pharmacological drugs of interest by automated, high throughput drug screening have become increasing relied upon in a variety of pharmaceutical and biotechnology drug development programs. Unfortunately, requisite drugs for such high throughput screening assays are not widespread. A significant reason for lack of progress in this area is insufficient understanding of molecules (i.e. effectors) that impact SREBP-1 and the LDL receptor.
It thus would be desirable to have anti-lipemic drugs with dual capacity to modulate the LDL receptor and serum cholesterol levels. It would be particularly desirable if such anti-lipemic drugs could be administered to subject mammal at doses near or below those presently used with many serum cholesterol inhibitors. It would be further desirable to have effective in vitro and in vivo assays for identifying drugs with potential to modulate the LDL receptor particularly involving SREBP-1 protein maturation.
The present invention generally relates to compositions and methods for modulating serum cholesterol in a subject mammal. In one aspect, the invention features novel anti-lipemic drugs that include at least one identified effector of the Low Density Lipoprotein (LDL) receptor and at least one identified serum cholesterol inhibitor. In a particular aspect, the drugs include one identified sphingolipid or protein modifying same linked to one identified serum cholesterol inhibitor. Additionally provided are methods for identifying anti-lipemic drugs capable of modulating the LDL receptor and specifically SREBP-1 maturation, including assays designed to identify pharmacological drugs capable of stabilizing or reducing serum cholesterol levels in a mammal and particularly a human patient.
We have discovered a wide spectrum of compositions and methods for treating or preventing disorders modulated by cholesterol. Sometimes the disorders will be referred to herein as xe2x80x9ccholesterol related disordersxe2x80x9d or a similar term. More specifically, we have identified anti-lipemic drugs that include at least one identified effector of the LDL receptor, and particularly an effector of SREBP-1 and at least one identified serum cholesterol inhibitor. Particular anti-lipemic drugs of this invention usually have one of each component although drugs having multiple effectors and inhibitors (e.g., between from about 2 to 5 of each) are contemplated. Preferred anti-lipemic drugs feature specifically defined characteristics such as capacity to stabilize or reduce serum cholesterol levels in a subject mammal as determined by in vitro or in vivo assays described below.
More specifically, the present invention provides a variety of specific anti-lipemic drugs and methods for using same for the treatment or prevention of one or more than one cholesterol related disorder in a subject mammal. Illustrative disorders are known in the field and include hyperlipoproteinemia including hypercholesterolemia, stroke, obesity, compulsive eating disorders, cardiac disease including atherosclerosis, cerebral atherosclerosis, cholesteryl ester storage disorder, liver disease including organ transplantation failure and cirrhosis; diseases of the biliary system, and viral infection, particularly those infections facilitating encephalitis or related disorders.
Particular anti-lipemic drugs in accord with this invention include one SREBP-1 effector and one synthetic or semi-synthetic inhibitor of an enzyme associated with cholesterol biosynthesis. Preferred enzymes have been extensively characterized and include 3-hydroxy-3-methylglutaryl (HMG) CoA reductase and HMG CoA synthetase. Additionally contemplated anti-lipemic drugs feature, as the effector component, an identified caspase, particularly the cpp32 protease (caspase-3), neutral sphingomyelinase (N-SMase), ceramide, SREBP-1 (precursor), or SREBP-1 (mature). Effective fragments of the N-SMase, cpp32 protease, SREBP-1 (precursor), or the SREBP-1 (mature) protein are contemplated as effector molecules within the scope of this invention.
Additionally specific anti-lipemic drugs include one effector of SREBP-1 which effector can be a sphingolipid, e.g., sphingomyelin or ceramide; or N-SMase or an effective fragment thereof. In embodiments in which the anti-lipemic drug includes ceramide, that ceramide molecule is preferably naturally-occurring (i.e., can be isolated in substantially pure formn from a biological source). A more preferred ceramide for use in the drug is any one of C-2, C-4, C-6 or C-8 ceramide. A preferred N-SMase molecule is encoded by specific nucleotide sequences disclosed herein including those encoding enzymatically active forms of that enzyme and effective fragments thereof. Preferred effectors in accord with this invention demonstrate substantial capacity to modulate the LDL receptor and especially maturation of the SREBP-1 protein as determined by specific assays described below.
As discussed, particular anti-lipemic drugs of this invention include a suitable SREBP-1 effector such as sphingolipid, particularly a sphingomyelin or ceramide, N-SMase or effective fragment thereof, although other drugs may include other effectors as needed. In this embodiment, the anti-lipemic drug further includes the inhibitor of HMG CoA reductase. It is generally preferred that the effector and the inhibitor are be combined in a way to facilitate function for which the drug was intended. A preferred function is to stabilize or reduce serum cholesterol as determined by a conventional in vivo assays defined below. In most instances, covalent attachment between the effector and the inhibitor will be preferred although other associations will be suitable for some applications. Preferred cholesterol inhibitors have recognized capacity to inhibit the reductase, thereby lowering serum cholesterol. Illustrative inhibitors include commercially available serum cholesterol inhibitors acceptable for human use, e.g., fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin), atorvastatin; or a clinically acceptable derivative thereof.
In a particular embodiment, the anti-lipemic drugs include one effector of the SREBP-1 protein, e.g., the N-SMase or effective fragment; or a sphingolipid. In this example, the effector is also preferably associated with the inhibitor of HMG CoA reductase. By the term xe2x80x9cassociatedxe2x80x9d or related term is meant that the SREBP-1 effector and the inhibitor are attached by at least one bond preferably at least on covalent bond. Particular examples of bonding are described below. In some instances, the association can also be provided by a suitable combination of covalent and non-covalent chemical bonds. Alternatively, association between the SREBP-1 effector and the inhibitor can be provided by essential co-administration of the effector and the inhibitor to a desired subject mammal. More specific methods for making and using the drugs of this invention are provided in the discussion and examples which follow.
In one embodiment, the anti-lipemic drug includes the sphingolipid attached to the inhibitor by at least one covalent bond. As noted, preferred are recognized cholesterol inhibitors such as fluvastatin, simvastatin, lovastatin, pravastatin, mevinolin (compactin), atorvastatin. In this illustration, the sphingolipid is preferably ceramide or a related molecule, particularly any one of the preferred ceramides described previously, which ceramide is covalently linked to a reactive hydroxyl group on the inhibitor molecule. Also in this example, the hydroxyl group of the inhibitor is usually covalently linked to a reactive carbon atom on the ceramide such as the C-3 carbon.
Additional anti-lipemic drugs of this invention include at least one bifunctional spacer group, typically a heterobifunctional spacer group, which group spaces the SREBP-1 effector from the inhibitor or other drug moiety. A particular example of this type of anti-lipemic drug includes one SREBP-1 effector covalently linked to one heterobifunctional spacer group. That spacer group is preferably covalently linked to the serum cholesterol inhibitor. Typically, the bifunctional spacer is linked to suitably reactive chemical group on the effector and the inhibitor, usually specifically reactive carbon atoms and hydroxyl groups, respectively.
Further anti-lipemic drugs in accord with the present invention include one effector of SREBP-1 such as the neutral sphingomyelinase (N-SMase) or an effective fragment thereof. A preferred drug includes the N-SMase or the fragment in association with an inhibitor of HMG CoA reductase or HMG CoA synthetase as described previously. Preferred examples of the N-SMase and fragment are provided in the examples and discussion which follow.
Further contemplated anti-lipemic drugs include the effector of SREBP-1, preferably the neutral sphingomyelinase (N-SMase) or the fragment thereof; which effector is covalently linked to one inhibitor of the HMG CoA reductase. Preferred inhibitors of the reductase have already been discussed. Preferably, the covalent linkage is made by binding a chemically reactive group on the enzyme or fragment, preferably an amide bond. More particular anti-lipemic drugs are disclosed below featuring an amide linkage between the enzyme or fragment and the serum cholesterol inhibitor.
Preferred anti-lipemic drugs of this invention are generally formulated to suit intended use and specifically include those drugs formatted for topical or related use. Additionally, the invention includes anti-lipemic drugs that include components sufficient to provide the drug as a liposome formulation suitable for in vitro or in vivo use. Methods for making and using such preferred drugs are described below.
In general, therapeutic methods in accord with this invention include administering to a subject, particularly a mammal such as a primate, especially a human, a therapeutically effective amount of at least one anti-lipemic drug of interest. That drug can be administered as a sole active agent. Alternatively, the anti-lipemic drug can be administered in combination with other drugs or agents exhibiting a desired pharmacological activity. In most cases, the amount of anti-lipemic drug use will be one which exhibits good activity in a standard in vitro or in vivo assay described below.
As discussed, the anti-lipemic drugs of this invention advantageously provide dual xe2x80x9canti-cholesterolxe2x80x9d activity, ie, by increasing LDL receptor activity, particularly by enhancing LDL receptor levels; and by reducing serum cholesterol levels. Particular in vitro and in vivo assays to detect and quantitate these activities are provided below and in the discussion and examples which follow.
As an illustration, preferred anti-lipemic drugs of this invention are capable of stimulating production of the mature form of SREBP-1 (maturation) by at least about 2 fold, as determined by a standard SREBP-1 proteolysis (maturation) assay. That assay is provided below and generally involves monitoring in a time and dose dependent manner, the maturation of the SREBP-1 protein. Mature SREBP-1 protein is believed to move to the nucleus and stimulate production of LDL receptor.
Additionally preferred anti-lipemic drugs of this invention are capable of increasing LDL receptor mRNA levels by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80, or 90% as determined by Northern blot or related mRNA detection assay. An exemplary Northern blot assay for detecting and optionally quantitating LDL receptor mRNA levels are provided below.
Also preferred anti-lipemic drugs of this invention exhibit an ID50 of between from about 20%, 30%, 40%, 50%, 60%, or 70% to about 90% as determined in a standard HMG CoA reductase assay. In this assay, the activity of the reductase enzyme is monitored in the presence and absence (control) of the anti-lipemic agent. An example of the standard HMG CoA reductase assay is provided below.
Further preferred anti-lipemic drugs are capable of significantly reducing serum cholesterol as determined by a standard serum cholesterol assay. Preferably, an administered anti-lipemic drug is capable of reducing serum cholesterol in a subject mammal by at least about 5%, 10% to 20% or 30%, 40%, 50%, 60% or 70%. An example of the assay is described below. Typically, the reduction in serum cholesterol is monitored with respect to a suitable control subject. The serum cholesterol assays are optimally performed in vivo and preferably include use of a recognized animal model such as specific rabbit and mouse strains provided below.
Preferred animal models for use in the serum cholesterol assay or other suitable assay disclosed herein are generally recognized test systems for an identified cholesterol related disease. Typically such animal models include commercially available in-bred strains of rabbits or mice, e.g., the Watanabe heritable hyperlipidemic rabbit and the apolipoprotein E negative mouse. In this example, the reduction in serum cholesterol can be evaluated using well-known testing strategies adopted for use with the specific animal model. However for some applications it may be useful to test a desired anti-lipemic drug on a normal (xe2x80x9cwild-typexe2x80x9d) animal such as those genetically defined (e.g., isogenic) wild-type animal strains known in the field.
The anti-lipemic drugs of this invention are preferably tested by at least one and preferably all of the standard assays summarized above. Preferred are anti-lipemic drugs that demonstrate about the stated activity ranges in one or more of the assays.
Significantly, use of multiple testing strategies (e.g., a combination of one in vitro and/or in vivo assays) with a single anti-lipemic drug can extend the selectivity and effectiveness of the testing as needed. That is, the testing strategy can be tailored for treatment or prevention of a particular cholesterol related disease or group of patients if required.
Such broad spectrum testing provides additional advantages. For example, preferred anti-lipemic drugs have capacity to enhance LDL receptor activity (typically by enhancing production of the LDL receptor) and provide for a reduction in serum cholesterol level. Thus by providing such dual xe2x80x9canti-cholesterolxe2x80x9d activity, the invention is a significant advance over prior therapies and agents that have been reported to reduce serum cholesterol in one way, usually by targeting cholesterol biosynthesis. Accordingly, preferred anti-lipemic drugs of this invention feature better activity, can be administered at lower dosages then prior agents. Patient tolerance of the anti-lipemic drugs will also be positively impacted.
In another aspect, the invention includes methods for modulating and particularly reducing serum cholesterol level in a mammal. In this embodiment, the methods generally include administering to the mammal a therapeutically effective amount of at least one and typically one of the anti-lipemic drugs disclosed herein.
Also provided are methods for modulating LDL receptor levels in a mammal in which the method includes administering to the mammal a therapeutically effective amount of at least one and typically one of the anti-lipemic drugs disclosed herein.
The present invention also provides methods treating a disorder in a mammal having or suspected of having high serum cholesterol levels. In this embodiment, the method includes administering to the mammal a therapeutically effective amount of at least one of the anti-lipemic drugs disclosed herein. A preferred mammal is a primate and especially a human patient, e.g., those susceptible to coronary heart disease, obesity, eating disorders or other cholesterol related disorders described herein. Accordingly, the methods are especially applicable to a subject mammal such as a human patient who has been diagnosed as having, is suspected of having, or is susceptible to a high serum cholesterol level, e.g., through adverse genetic or dietary influences.
Also provided by this invention are methods for modulating serum cholesterol level in a mammal in which the method includes administering to the mammal a therapeutically effective amount of at least one of the anti-lipemic drugs disclosed herein. In this embodiment, the SREBP-1 effector is preferably neutral shpingomyelinase (N-SMase) or an effective fragment thereof; or a sphingolipid such as cermide. Preferred methods employ a primate such as a human patient. Preferred anti-lipemic agents for use in the methods are typically tested for activity using a recognized animal model for a cholesterol related disorder and especially atherosclerosis, e.g., the Watanabe heritable hyperlipidemic rabbit or an apolipoprotein E negative mouse discussed previously.
Additionally contemplated are methods for modulating LDL receptor in a mammal in which the methods include administering to the mammal a therapeutically effective amount of at least one of the anti-lipemic drugs disclosed herein. The modulation is preferably an increase in the synthesis (or sometimes decrease in the degradation of) the LDL receptor. In this example, the SREBP-1 effector is neutral sphingomyelinase (N-SMase) or an effective fragment thereof; or a sphingolipid such as ceramide. Methods for evaluating an increase or decrease in LDL receptor levels are known in the field and involve, e.g., molecular and immunological approaches using anti-LDL antibodies capable of detecting and quantitating LDL receptor in vitro or in vivo.
Particular methods of this invention involve use of at least one suitable anti-lipemic drug which includes one effector of SREBP-1 associated with an identified inhibitor of serum cholesterol as discussed herein. In this example, that effector is preferably a sphingolipid such as ceramide. Preferred examples of ceramide include naturally occurring ceramide and other ceramide forms as discussed previously. As discussed, preferred methods are conducted using a mammalian subject such as a primate and especially a human patient who has been diagnosed as having, is suspected of having, or is susceptible to a cholesterol related disorder as disclosed.
In an embodiment of the methods disclosed herein, the anti-lipemic drug is preferably disposed as a liposome formulation. In this example, the liposome formation can be compatible for hepatic administration in accordance with standard practice. Also in this example, the liposome formulation can be administered to the liver or associated organ in a human patient according to standard medical techniques involving, e.g., oral, intramuscular, intraperitoneal, administration via a stent or related implementation. Particular routes of administration are provided below.
Other aspects of the invention are discussed below.